3, 10, and 12a substituted tetracycline compounds

ABSTRACT

The present invention pertains to novel 3, 10, and/or 12a-substituted tetracycline compounds. These tetracycline compounds can be used to treat numerous tetracycline compound-responsive states, such as bacterial infections and neoplasms, as well as other known applications for minocycline and tetracycline compounds in general, such as blocking tetracycline efflux and modulation of gene expression.

RELATED APPLICATION

This application is a continuation application of U.S. Ser. No.10/853,537, entitled “3, 10 and 12a Substituted Tetracycline Compounds”filed on May 24, 2004; which is a continuation application of U.S. Ser.No. 10/619,653, entitled “3, 10 and 12a Substituted TetracyclineCompounds” filed on Jul. 14, 2003; which claims priority to U.S.Provisional Patent Application Ser. No. 60/395,696, entitled “3, 10 and12a Substituted Tetracycline Compounds” filed on Jul. 12, 2002, theentire contents of each of the aforementioned applications are herebyincorporated herein by reference.

BACKGROUND OF THE INVENTION

The development of the tetracycline antibiotics was the direct result ofa systematic screening of soil specimens collected from many parts ofthe world for evidence of microorganisms capable of producingbacteriocidal and/or bacteriostatic compositions. The first of thesenovel compounds was introduced in 1948 under the name chlortetracycline.Two years later, oxytetracycline became available. The elucidation ofthe chemical structure of these compounds confirmed their similarity andfurnished the analytical basis for the production of a third member ofthis group in 1952, tetracycline. A new family of tetracyclinecompounds, without the ring-attached methyl group present in earliertetracyclines, was prepared in 1957 and became publicly available in1967; and minocycline was in use by 1972.

Recently, research efforts have focused on developing new tetracyclineantibiotic compositions effective under varying therapeutic conditionsand routes of administration. New tetracycline analogues have also beeninvestigated which may prove to be equal to or more effective than theoriginally introduced tetracycline compounds. Examples include U.S. Pat.Nos. 2,980,584; 2,990,331; 3,062,717; 3,165,531; 3,454,697; 3,557,280;3,674,859; 3,957,980; 4,018,889; 4,024,272; and 4,126,680. These patentsare representative of the range of pharmaceutically active tetracyclineand tetracycline analogue compositions.

Historically, soon after their initial development and introduction, thetetracyclines were found to be highly effective pharmacologicallyagainst rickettsiae; a number of gram-positive and gram-negativebacteria; and the agents responsible for lymphogranuloma venereum,inclusion conjunctivitis, and psittacosis. Hence, tetracyclines becameknown as “broad spectrum” antibiotics. With the subsequent establishmentof their in vitro antimicrobial activity, effectiveness in experimentalinfections, and pharmacological properties, the tetracyclines as a classrapidly became widely used for therapeutic purposes. However, thiswidespread use of tetracyclines for both major and minor illnesses anddiseases led directly to the emergence of resistance to theseantibiotics even among highly susceptible bacterial species bothcommensal and pathogenic (e.g., pneumococci and Salmonella). The rise oftetracycline-resistant organisms has resulted in a general decline inuse of tetracyclines and tetracycline analogue compositions asantibiotics of choice.

SUMMARY OF THE INVENTION

In one embodiment, the invention pertains, at least in part, totetracycline compounds of Formula I:

wherein:

X is CHC(R¹³Y′Y), CR^(6′)R⁶, S, NR⁶, or O;

R^(2″) is —C(═O)NR²R^(2′), or —CN;

R², R^(2′), R^(4a), and R^(4b) are each independently hydrogen, alkyl,alkenyl, alkynyl, alkoxy, alkylthio, alkylsulfinyl, alkylsulfonyl,alkylamino, arylalkyl, aryl, heterocyclic, heteroaromatic or a prodrugmoiety;

R³, R¹⁰, R¹¹, R¹², and R^(12″) are each independently hydrogen, alkyl,alkenyl, aryl, alkynyl, aralkyl, acetyl, alkylcarbonyl, alkenylcarbonyl,arylcarbonyl, alkynylcarbonyl, alkyloxycarbonyl, alkenyloxycarbonyl,alkynyloxycarbonyl, aryloxycarbonyl, alkylaminocarbonyl,alkenylaminocarbonyl, alkynylaminocarbonyl, arylaminocarbonyl,alkylthiocarbonyl, alkenylthiocarbonyl, alkynylthiocarbonyl,arylthiocarbonyl, alkyloxythiocarbonyl, alkenyloxythiocarbonyl,alkynyloxythiocarbonyl, aryloxythiocarbonyl, alkylaminothiocarbonyl,alkenylaminothiocarbonyl, alkynylaminothiocarbonyl,arylaminothiocarbonyl, alkylthiothiocarbonyl, alkenylthiothiocarbonyl,alkynylthiothiocarbonyl, arylthiothiocarbonyl, provided that at leastone of R³, R¹⁰, R¹¹, or R¹² is not hydrogen when R² is —C(═O)NR²R^(2′);

R⁴ and R^(4′) are each independently NR^(4a)R^(4b), alkyl, alkenyl,alkynyl, hydroxyl, halogen, hydrogen, or when taken together the oxygenof a carbonyl group;

R⁵ is hydroxyl, hydrogen, thiol, alkanoyl, aroyl, alkaroyl, aryl,heteroaromatic, alkyl, alkenyl, alkynyl, alkoxy, alkylthio,alkylsulfinyl, alkylsulfonyl, alkylamino, arylalkyl, alkyl carbonyloxy,or aryl carbonyloxy;

R⁶ and R^(6′) are each independently hydrogen, methylene, absent,hydroxyl, halogen, thiol, alkyl, alkenyl, alkynyl, aryl, alkoxy,alkylthio, alkylsulfinyl, alkylsulfonyl, alkylamino, or an arylalkyl;

R⁷ is hydrogen, hydroxyl, halogen, thiol, nitro, alkyl, alkenyl,alkynyl, aryl, alkoxy, alkylthio, alkylsulfinyl, alkylsulfonyl,arylalkyl, amino, arylalkenyl, arylalkynyl, acyl, aminoalkyl,heterocyclic, thionitroso, or —(CH₂)₀₋₃NR^(7c)C(═W′)WR^(7a);

R⁸ is hydrogen, hydroxyl, halogen, thiol, nitro, alkyl, alkenyl,alkynyl, aryl, alkoxy, alkylthio, alkylsulfinyl, alkylsulfonyl,alkylamino, amino, arylalkenyl, arylalkynyl, acyl, aminoalkyl,heterocyclic, thionitroso, or —(CH₂)₀₋₃NR^(8c)C(=E′)ER^(8a);

R⁹ is hydrogen, hydroxyl, halogen, thiol, nitro, alkyl, alkenyl,alkynyl, aryl, alkoxy, alkylthio, alkylsulfinyl, alkylsulfonyl,arylalkyl, amino, arylalkenyl, arylalkynyl, acyl, aminoalkyl,heterocyclic, thionitroso, —(CH₂)₀₋₃NR^(9c)C(=Z′)ZR^(9a), or R⁹ isoptionally linked to R¹⁰ to form a heterocyclic ring;

R^(7a), R^(7b), R^(7c), R^(7d), R^(7e), R^(7f), R^(8a), R^(8b), R^(8c),R^(8d), R^(8e), R^(8f), R^(9a), R^(9b), R^(9c), R^(9d), R^(9e), andR^(8f) are each independently hydrogen, acyl, alkyl, alkenyl, alkynyl,alkoxy, alkylthio, alkylsulfinyl, alkylsulfonyl, alkylamino, arylalkyl,aryl, heterocyclic, heteroaromatic or a prodrug moiety;

R^(12′) is OR¹² or NR¹²R^(12″);

R¹³ is hydrogen, hydroxy, alkyl, alkenyl, alkynyl, alkoxy, alkylthio,aryl, alkylsulfinyl, alkylsulfonyl, alkylamino, or an arylalkyl;

E is CR^(8d)R^(8e), S, NR^(8b) or O;

E′ is O, NR^(8f), or S;

W is CR^(7d)R^(7e), S, NR^(7b) or O;

W′ is O, NR^(7f), or S;

X is CHC(R¹³Y′Y), C═CR¹³Y, CR^(6′)R⁶, S, NR⁶, or O;

Y′ and Y are each independently hydrogen, halogen, hydroxyl, cyano,sulfhydryl, amino, alkyl, alkenyl, alkynyl, alkoxy, alkylthio,alkylsulfinyl, alkylsulfonyl, alkylamino, or an arylalkyl;

Z is CR^(9d)R^(9e), S, NR^(9b) or O;

Z′ is O, S, or NR^(9f), and pharmaceutically acceptable salts, estersand enantiomers thereof.

The invention also pertains, at least in part, to methods forderivatizing tetracycline compounds at the 3-position, 10, position,and/or 12a position. The methods include contacting a tetracyclinecompound with an appropriate base under appropriate first conditions,and contacting the tetracycline compound with a derivatizing reagentunder appropriate second conditions.

The invention also pertains, at least in part, to methods fordederivatizing a derivatized tetracycline compound (e.g., a tetracyclinecompound derivatized at the 3-position, 10-position, and/or12a-position). The method includes contacting the derivatizedtetracycline compound with an appropriate dederivatizing agent underappropriate conditions, such that derivatized tetracycline compound isdederivatized.

In another embodiment, the invention pertains, at least in part, tomethods for treating a tetracycline responsive state in a subject, byadministering to the subject a tetracycline compound of the invention.

In a further embodiment, the invention also pertains to pharmaceuticalcompositions, which contain a therapeutically effective amount of atetracycline compound of the invention or mixtures thereof, and apharmaceutically acceptable carrier.

DETAILED DESCRIPTION OF THE INVENTION

The invention pertains, at least in part, to tetracycline compounds ofFormula I:

wherein:

X is CHC(R¹³Y′Y), CR^(6′)R⁶, S, NR⁶, or O;

R^(2″) is —C(═O)NR²R^(2′), or —CN;

R², R^(2′), R^(4a), and R^(4b) are each independently hydrogen, alkyl,alkenyl, alkynyl, alkoxy, alkylthio, alkylsulfinyl, alkylsulfonyl,alkylamino, arylalkyl, aryl, heterocyclic, heteroaromatic or a prodrugmoiety;

R³, R¹⁰, R¹¹, R¹², and R^(12″) are each independently hydrogen, alkyl,alkenyl, aryl, alkynyl, aralkyl, acetyl, alkylcarbonyl, alkenylcarbonyl,arylcarbonyl, alkynylcarbonyl, alkyloxycarbonyl, alkenyloxycarbonyl,alkynyloxycarbonyl, aryloxycarbonyl, alkylaminocarbonyl,alkenylaminocarbonyl, alkynylaminocarbonyl, arylaminocarbonyl,alkylthiocarbonyl, alkenylthiocarbonyl, alkynylthiocarbonyl,arylthiocarbonyl, alkyloxythiocarbonyl, alkenyloxythiocarbonyl,alkynyloxythiocarbonyl, aryloxythiocarbonyl, alkylaminothiocarbonyl,alkenylaminothiocarbonyl, alkynylaminothiocarbonyl,arylaminothiocarbonyl, alkylthiothiocarbonyl, alkenylthiothiocarbonyl,alkynylthiothiocarbonyl, arylthiothiocarbonyl, provided that at leastone of R³, R¹⁰, R¹¹, or R¹² is not hydrogen when R² is —C(═O)NR²R^(2′);

R⁴ and R^(4′) are each independently NR^(4a)R^(4b), alkyl, alkenyl,alkynyl, hydroxyl, halogen, hydrogen, or when taken together the oxygenof a carbonyl group;

R⁵ is hydroxyl, hydrogen, thiol, alkanoyl, aroyl, alkaroyl, aryl,heteroaromatic, alkyl, alkenyl, alkynyl, alkoxy, alkylthio,alkylsulfinyl, alkylsulfonyl, alkylamino, arylalkyl, alkyl carbonyloxy,or aryl carbonyloxy;

R⁶ and R^(6′) are each independently hydrogen, methylene, absent,hydroxyl, halogen, thiol, alkyl, alkenyl, alkynyl, aryl, alkoxy,alkylthio, alkylsulfinyl, alkylsulfonyl, alkylamino, or an arylalkyl;

R⁷ is hydrogen, hydroxyl, halogen, thiol, nitro, alkyl, alkenyl,alkynyl, aryl, alkoxy, alkylthio, alkylsulfinyl, alkylsulfonyl,arylalkyl, amino, arylalkenyl, arylalkynyl, acyl, aminoalkyl,heterocyclic, thionitroso, or —(CH₂)₀₋₃NR^(7c)C(═W′)WR^(7a);

R⁸ is hydrogen, hydroxyl, halogen, thiol, nitro, alkyl, alkenyl,alkynyl, aryl, alkoxy, alkylthio, alkylsulfinyl, alkylsulfonyl,alkylamino, amino, arylalkenyl, arylalkynyl, acyl, aminoalkyl,heterocyclic, thionitroso, or —(CH₂)₀₋₃NR^(8c)C(=E′)ER^(8a);

R⁹ is hydrogen, hydroxyl, halogen, thiol, nitro, alkyl, alkenyl,alkynyl, aryl, alkoxy, alkylthio, alkylsulfinyl, alkylsulfonyl,arylalkyl, amino, arylalkenyl, arylalkynyl, acyl, aminoalkyl,heterocyclic, thionitroso, or —(CH₂)₀₋₃NR^(9c)C(=Z′)ZR^(9a);

R^(7a), R^(7b), R^(7c), R^(7d), R^(7e), R^(7f), R^(8a), R^(8b), R^(8c),R^(8d), R^(8e), R^(8f), R^(9a), R^(9b), R^(9c), R^(9d), R^(9e), andR^(8f) are each independently hydrogen, acyl, alkyl, alkenyl, alkynyl,alkoxy, alkylthio, alkylsulfinyl, alkylsulfonyl, alkylamino, arylalkyl,aryl, heterocyclic, heteroaromatic or a prodrug moiety;

R^(12′) is OR¹² or NR¹²R^(12″);

R¹³ is hydrogen, hydroxy, alkyl, alkenyl, alkynyl, alkoxy, alkylthio,aryl, alkylsulfinyl, alkylsulfonyl, alkylamino, or an arylalkyl;

E is CR^(8d)R^(8e), S, NR^(8b) or O;

E′ is O, NR^(8f), or S;

W is CR^(7d)R^(7e), S, NR^(7b) or O;

W′ is O, NR^(7f), or S;

X is CHC(R¹³Y′Y), C═CR¹³Y, CR^(6′)R⁶, S, NR⁶, or O;

Y′ and Y are each independently hydrogen, halogen, hydroxyl, cyano,sulfhydryl, amino, alkyl, alkenyl, alkynyl, alkoxy, alkylthio,alkylsulfinyl, alkylsulfonyl, alkylamino, or an arylalkyl;

Z is CR^(9d)R^(9e), S, NR^(9b) or O;

Z′ is O, S, or NR^(9f), and pharmaceutically acceptable salts, estersand enantiomers thereof.

The term “tetracycline compound” includes many compounds with a similarring structure to tetracycline. Examples of tetracycline compoundsinclude: tetracycline, oxytetracycline, chlortetracycline,demeclocycline, doxycycline, chelocardin, minocycline, rolitetracycline,lymecycline, sancycline, methacycline, apicycline, clomocycline,guamecycline, meglucycline, mepylcycline, penimepicycline, pipacycline,etamocycline, and penimocycline. Other derivatives and analoguescomprising a similar four ring structure are also included. The termincludes 4-dedimethylamino derivatives. Table 1 depicts tetracycline andseveral known tetracycline derivatives. The tetracycline compounds maybe unsubstituted at any position or further substituted, for example, atthe 2, 4, 5, 6, 7, 8, 9, or 13 position of the ring.

TABLE I

Tetracycline

Methacycline

Oxytetracycline

Doxycycline

The term “substituted tetracycline compounds” includes tetracyclinecompounds with substitution at the 3, 10, and/or 12a-position of thetetracycline molecule other than hydroxy. In one embodiment, thesubstitution enhances the ability of the tetracycline compound toperform its intended function, e.g., treat tetracycline responsivestates. In an embodiment, the substituted tetracycline compound issubstituted tetracycline (e.g., wherein R⁴ is NR^(4a)R^(4b); R^(4a) andR^(4b) are methyl, R⁵ is hydrogen and X is CR⁶R^(6′), wherein R⁶ ismethyl and R^(6′) is hydroxy); substituted doxycycline (e.g., wherein R⁴is NR^(4a)R^(4b); R^(4a) and R^(4b) are methyl, R⁵ is hydroxyl and X isCR⁶R^(6′), wherein R⁶ is methyl and R^(6′) is hydrogen); or substitutedsancycline (wherein R⁴ is NR^(4a)R^(4b); R^(4a) and R^(4b) are methyl;R⁵ is hydrogen and X is CR⁶R^(6′) wherein R⁶ and R^(6′) are hydrogenatoms. In one embodiment, R⁴ and R^(4′) are each hydrogen or the oxygenof a carbonyl group. In an embodiment, the compounds of the invention donot include compounds wherein R¹⁰ is a sugar or compounds described inDD 268 951, incorporated herein by reference in its entirety.

The tetracycline compounds of the invention include, for example,compounds wherein X is CR⁶R^(6′); R⁴ and R^(4′) are hydrogen or theoxygen of a carbonyl group; R², R^(2′), R⁶, R^(6′), R⁸, R¹⁰, R¹¹, andR¹² are each hydrogen; R^(4a) and R^(4b) are lower alkyl; and R⁵ ishydroxy or hydrogen. In an embodiment, R⁴ and R^(4′) are each hydrogenand R⁵ is hydrogen. In a further embodiment, the compounds of theinvention include compounds wherein R⁴ is dialkylamino, R^(4′) ishydrogen and R⁵ is hydrogen.

In one embodiment, the tetracycline compounds of the invention includetetracycline compounds of formula (I), wherein R³ is alkyl, alkenyl,aryl, acetyl, aralkyl, alkynyl, alkylcarbonyl, alkenylcarbonyl,arylcarbonyl, alkynylcarbonyl, alkyloxycarbonyl, alkenyloxycarbonyl,alkynyloxycarbonyl, aryloxycarbonyl, alkylaminocarbonyl,alkenylaminocarbonyl, alkynylaminocarbonyl, arylaminocarbonyl,alkylthiocarbonyl, alkenylthiocarbonyl, alkynylthiocarbonyl,arylthiocarbonyl, alkyloxythiocarbonyl, alkenyloxythiocarbonyl,alkynyloxythiocarbonyl, aryloxythiocarbonyl, alkylaminothiocarbonyl,alkenylaminothiocarbonyl, alkynylaminothiocarbonyl,arylaminothiocarbonyl, alkylthiothiocarbonyl, alkenylthiothiocarbonyl,alkynylthiothiocarbonyl, or arylthiothiocarbonyl.

In a further embodiment, R³ is aralkyl, e.g., benzyl. In another furtherembodiment, R³ is alkyl, alkenyl, or acetyl. In another furtherembodiment (e.g., when R¹⁰ and R^(12a) are not both hydrogen), R³ ishydrogen.

In one embodiment, the tetracycline compounds of the invention includetetracycline compounds of formula (I), wherein R¹⁰ is alkyl, alkenyl,aryl, acetyl, aralkyl, alkynyl, alkylcarbonyl, alkenylcarbonyl,arylcarbonyl, alkynylcarbonyl, alkyloxycarbonyl, alkenyloxycarbonyl,alkynyloxycarbonyl, aryloxycarbonyl, alkylaminocarbonyl,alkenylaminocarbonyl, alkynylaminocarbonyl, arylaminocarbonyl,alkylthiocarbonyl, alkenylthiocarbonyl, alkynylthiocarbonyl,arylthiocarbonyl, alkyloxythiocarbonyl, alkenyloxythiocarbonyl,alkynyloxythiocarbonyl, aryloxythiocarbonyl, alkylaminothiocarbonyl,alkenylaminothiocarbonyl, alkynylaminothiocarbonyl,arylaminothiocarbonyl, alkylthiothiocarbonyl, alkenylthiothiocarbonyl,alkynylthiothiocarbonyl, or arylthiothiocarbonyl.

In a further embodiment, R¹⁰ is aralkyl, e.g., benzyl. In anotherfurther embodiment, R¹⁰ is alkyl, alkenyl, or acetyl. In another furtherembodiment (e.g., when R³ and R¹² are not both hydrogen), R¹⁰ ishydrogen.

In one embodiment, the tetracycline compounds of the invention includetetracycline compounds of formula (I), wherein R¹² is alkyl, alkenyl,aryl, acetyl, aralkyl, alkynyl, alkylcarbonyl, alkenylcarbonyl,arylcarbonyl, alkynylcarbonyl, alkyloxycarbonyl, alkenyloxycarbonyl,alkynyloxycarbonyl, aryloxycarbonyl, alkylaminocarbonyl,alkenylaminocarbonyl, alkynylaminocarbonyl, arylaminocarbonyl,alkylthiocarbonyl, alkenylthiocarbonyl, alkynylthiocarbonyl,arylthiocarbonyl, alkyloxythiocarbonyl, alkenyloxythiocarbonyl,alkynyloxythiocarbonyl, aryloxythiocarbonyl, alkylaminothiocarbonyl,alkenylaminothiocarbonyl, alkynylaminothiocarbonyl,arylaminothiocarbonyl, alkylthiothiocarbonyl, alkenylthiothiocarbonyl,alkynylthiothiocarbonyl, or arylthiothiocarbonyl.

In a further embodiment, R¹² is aralkyl, e.g., benzyl. In anotherfurther embodiment, R¹² is alkyl, alkenyl, or acetyl. In another furtherembodiment (e.g., when R³ and R¹⁰ are not both hydrogen), R¹² ishydrogen.

In one embodiment, R^(2″) is cyano.

In another embodiment, R^(12′) is NR¹²R^(12″). R¹² may be alkyl andR^(12″) may be hydrogen.

Examples of compounds of the invention include, but are not limited to:

In another embodiment, the invention pertains to methods forderivatizing tetracycline compounds at least at the 3-position, the10-position, and/or the 12a-position. The method includes contacting atetracycline compound with an appropriate base under appropriate firstconditions, and contacting the tetracycline compound with a derivatizingreagent under appropriate second conditions, such that said tetracyclinecompound is derivatized. The 3-position, 10-position and 12a-positionare shown on the tetracycline structure below.

The term “derivatizing” includes changing the substituents at the 3-,10-, and/or 12a position to a substituent other than hydroxyl. Thetetracycline compound may be derivatized by contacting it with anappropriate base under appropriate conditions.

Examples of appropriate bases include bases which are capable ofdeprotonating the 3-, 10-, and/or 12a hydroxyl groups. Examples of suchbases include sodium hydride. Preferably, the base is selectivelydeprotonates the tetracycline compound at a particular position, suchthat the resulting derivatized tetracycline compound is relatively freeof positional isomers. Other bases which may be used include potassiumhydride and sodium hydroxide.

The term “appropriate first conditions” include conditions in which thebase can interact with the tetracycline compound, and deprotonate thedesired hydroxyl group. In one embodiment, the appropriate firstconditions comprise an aprotic solvent. Examples of suitable solventsinclude dimethylformamide (DMF), and other aprotic solvents. Theappropriate first conditions may also comprise altering the temperatureof the reaction components. In certain embodiments, the tetracyclinecompound and the appropriate base are allowed to react at roomtemperature for an appropriate length of time. One of ordinary skill inthe art will be able to use art recognized techniques to determine thelength of time needed to allow the reaction to near completion.Preferably, the appropriate first conditions are selected such that thesolvent and the atmosphere are inert to the tetracycline compound andbase.

The term “derivatizing agent” includes agents which can react with thetetracycline compound (after being treated with an appropriate base), toform a derivatized tetracycline compound (e.g., a tetracycline compoundwith a substituent at the 3, 10, or 12a position). Examples ofderivatizing agents include, but are not limited to, alkyl halides,alkenyl halides, alkynyl halides, acid halides, isocyanates,isothiocyanates, esters, carbonates, silyl halides (e.g., silylchlorides), ketones, aldehydes, Mannich bases, diazonium compounds, andtheir salts.

The term “appropriate second conditions” include conditions which allowderivatizing agents to interact with tetracycline compounds (optionallydeprotonated) to form derivatized tetracycline compounds. Preferably,the appropriate first conditions are selected such that the conditions(including, for example, the solvent and the atmosphere) are inert tothe derivatizing agent, the tetracycline compound, and the base. Incertain embodiments, the appropriate second conditions may be the same,different, or partially the same and partially different than theappropriate first conditions. For example, in certain embodiments, theappropriate second conditions may comprise heating the reaction mixture.An ordinarily skilled artisan will be able to determine using artrecognized techniques the appropriate length of time and temperature forthe reaction to take place.

In a further embodiment, the invention pertains to a method fordederivatizing a derivatized tetracycline compound (e.g., a tetracyclinecompound derivatized at the 3-position, 10-position, and/or12a-position). The method includes contacting a derivatized tetracyclinecompound with an appropriate dederivatizing agent under appropriateconditions, such that derivatized tetracycline compound isdederivatized.

Examples of dederivatizing agents include agents which can alter thederivatized tetracycline compounds to tetracycline compound without thederivatization at one or more positions of the tetracycline molecule.Examples of such agents include, for example, acids, or other hydrolysisor deprotection methods known in the art. In certain embodiments,substituents at position 3, 10, and/or 12a are hydroxyl or O— afterbeing treated with a dederivatizing agent under appropriate conditions.

The term “appropriate conditions” include reaction conditions (e.g.,atmosphere, solvent, temperature, time, etc.) which allow the reactionto take place. An ordinarily skilled artisan will be able to manipulatethe reaction conditions to obtain a desired result.

In certain embodiments, the tetracycline compounds may be dederivatizedafter altering one or more substituents on the tetracycline compound.The substituents which may be altered include any substituent which canbe altered on the tetracycline compound. Examples of such reactionswhich can be done on the derivatized or underivatized tetracyclinecompound include those described in, for example, U.S. Ser. No.09/895,857; U.S. Ser. No. 09/895,812; U.S. Pat. No. 5,326,759; U.S. Pat.No. 5,328,902; U.S. Pat. No. 5,495,031; U.S. Pat. No. 5,495,018; U.S.Pat. No. 5,495,030; U.S. Pat. No. 5,495,032; U.S. Pat. No. 5,512,553;U.S. Pat. No. 5,675,030; U.S. Pat. No. 5,843,925; U.S. Pat. No.5,886,175; U.S. Pat. No. 6,165,999; U.S. Pat. No. 3,239,499; WO95/22529; U.S. Pat. No. 5,064,821; U.S. Pat. No. 5,589,470; and U.S.Pat. No. 5,811,412, all incorporated herein by reference. In anembodiment, the present invention also pertains to 3, 10, and/or 12aderivatives of each of the tetracycline compounds disclosed in each ofthe above references.

The term “alkyl” includes saturated aliphatic groups, includingstraight-chain alkyl groups (e.g., methyl, ethyl, propyl, butyl, pentyl,hexyl, heptyl, octyl, nonyl, decyl, etc.), branched-chain alkyl groups(isopropyl, tert-butyl, isobutyl, etc.), cycloalkyl (alicyclic) groups(cyclopropyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl), alkylsubstituted cycloalkyl groups, and cycloalkyl substituted alkyl groups.The term alkyl further includes alkyl groups, which can further includeoxygen, nitrogen, sulfur or phosphorous atoms replacing one or morecarbons of the hydrocarbon backbone. In certain embodiments, a straightchain or branched chain alkyl has 6 or fewer carbon atoms in itsbackbone (e.g., C₁-C₆ for straight chain, C₃-C₆ for branched chain), andmore preferably 4 or fewer. Likewise, preferred cycloalkyls have from3-8 carbon atoms in their ring structure, and more preferably have 5 or6 carbons in the ring structure. The term C₁-C₆ includes alkyl groupscontaining 1 to 6 carbon atoms.

Moreover, the term alkyl includes both “unsubstituted alkyls” and“substituted alkyls”, the latter of which refers to alkyl moietieshaving substituents replacing a hydrogen on one or more carbons of thehydrocarbon backbone. Such substituents can include, for example,alkenyl, alkynyl, halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy,alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl,arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl,dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate,phosphonato, phosphinato, cyano, amino (including alkyl amino,dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino(including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido),amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate,sulfates, alkylsulfinyl, sulfonato, sulfamoyl, sulfonamido, nitro,trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an aromaticor heteroaromatic moiety. Cycloalkyls can be further substituted, e.g.,with the substituents described above. An “alkylaryl” or an “arylalkyl”moiety is an alkyl substituted with an aryl (e.g., phenylmethyl(benzyl)). The term “alkyl” also includes the side chains of natural andunnatural amino acids.

The term “aryl” includes groups, including 5- and 6-membered single-ringaromatic groups that may include from zero to four heteroatoms, forexample, benzene, phenyl, pyrrole, furan, thiophene, thiazole,isothiaozole, imidazole, triazole, tetrazole, pyrazole, oxazole,isooxazole, pyridine, pyrazine, pyridazine, and pyrimidine, and thelike. Furthermore, the term “aryl” includes multicyclic aryl groups,e.g., tricyclic, bicyclic, e.g., naphthalene, benzoxazole,benzodioxazole, benzothiazole, benzoimidazole, benzothiophene,methylenedioxophenyl, quinoline, isoquinoline, naphthridine, indole,benzofuran, purine, benzofuran, deazapurine, or indolizine. Those arylgroups having heteroatoms in the ring structure may also be referred toas “aryl heterocycles”, “heterocycles,” “heteroaryls” or“heteroaromatics”. The aromatic ring can be substituted at one or morering positions with such substituents as described above, as forexample, halogen, hydroxyl, alkoxy, alkylcarbonyloxy, arylcarbonyloxy,alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl,alkylaminoacarbonyl, arylalkyl aminocarbonyl, alkenylaminocarbonyl,alkylcarbonyl, arylcarbonyl, arylalkylcarbonyl, alkenylcarbonyl,alkoxycarbonyl, aminocarbonyl, alkylthiocarbonyl, phosphate,phosphonato, phosphinato, cyano, amino (including alkyl amino,dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino(including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido),amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate,sulfates, alkylsulfinyl, sulfonato, sulfamoyl, sulfonamido, nitro,trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an aromaticor heteroaromatic moiety. Aryl groups can also be fused or bridged withalicyclic or heterocyclic rings which are not aromatic so as to form apolycycle (e.g., tetralin).

The term “alkenyl” includes unsaturated aliphatic groups analogous inlength and possible substitution to the alkyls described above, but thatcontain at least one double bond.

For example, the term “alkenyl” includes straight-chain alkenyl groups(e.g., ethylenyl, propenyl, butenyl, pentenyl, hexenyl, heptenyl,octenyl, nonenyl, decenyl, etc.), branched-chain alkenyl groups,cycloalkenyl (alicyclic) groups (cyclopropenyl, cyclopentenyl,cyclohexenyl, cycloheptenyl, cyclooctenyl), alkyl or alkenyl substitutedcycloalkenyl groups, and cycloalkyl or cycloalkenyl substituted alkenylgroups. The term alkenyl further includes alkenyl groups which includeoxygen, nitrogen, sulfur or phosphorous atoms replacing one or morecarbons of the hydrocarbon backbone. In certain embodiments, a straightchain or branched chain alkenyl group has 6 or fewer carbon atoms in itsbackbone (e.g., C₂-C₆ for straight chain, C₃-C₆ for branched chain).Likewise, cycloalkenyl groups may have from 3-8 carbon atoms in theirring structure, and more preferably have 5 or 6 carbons in the ringstructure. The term C₂-C₆ includes alkenyl groups containing 2 to 6carbon atoms.

Moreover, the term alkenyl includes both “unsubstituted alkenyls” and“substituted alkenyls”, the latter of which refers to alkenyl moietieshaving substituents replacing a hydrogen on one or more carbons of thehydrocarbon backbone. Such substituents can include, for example, alkylgroups, alkynyl groups, halogens, hydroxyl, alkylcarbonyloxy,arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate,alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aminocarbonyl,alkylaminocarbonyl, dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl,phosphate, phosphonato, phosphinato, cyano, amino (including alkylamino, dialkylamino, arylamino, diarylamino, and alkylarylamino),acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyland ureido), amidino, imino, sulfhydryl, alkylthio, arylthio,thiocarboxylate, sulfates, alkylsulfinyl, sulfonato, sulfamoyl,sulfonamido, nitro, trifluoromethyl, cyano, azido, heterocyclyl,alkylaryl, or an aromatic or heteroaromatic moiety.

The term “alkynyl” includes unsaturated aliphatic groups analogous inlength and possible substitution to the alkyls described above, butwhich contain at least one triple bond.

For example, the term “alkynyl” includes straight-chain alkynyl groups(e.g., ethynyl, propynyl, butynyl, pentynyl, hexynyl, heptynyl, octynyl,nonynyl, decynyl, etc.), branched-chain alkynyl groups, and cycloalkylor cycloalkenyl substituted alkynyl groups. The term alkynyl furtherincludes alkynyl groups which include oxygen, nitrogen, sulfur orphosphorous atoms replacing one or more carbons of the hydrocarbonbackbone. In certain embodiments, a straight chain or branched chainalkynyl group has 6 or fewer carbon atoms in its backbone (e.g., C₂-C₆for straight chain, C₃-C₆ for branched chain). The term C₂-C₆ includesalkynyl groups containing 2 to 6 carbon atoms.

Moreover, the term alkynyl includes both “unsubstituted alkynyls” and“substituted alkynyls”, the latter of which refers to alkynyl moietieshaving substituents replacing a hydrogen on one or more carbons of thehydrocarbon backbone. Such substituents can include, for example, alkylgroups, alkynyl groups, halogens, hydroxyl, alkylcarbonyloxy,arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate,alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aminocarbonyl,alkylaminocarbonyl, dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl,phosphate, phosphonato, phosphinato, cyano, amino (including alkylamino, dialkylamino, arylamino, diarylamino, and alkylarylamino),acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyland ureido), amidino, imino, sulfhydryl, alkylthio, arylthio,thiocarboxylate, sulfates, alkylsulfinyl, sulfonato, sulfamoyl,sulfonamido, nitro, trifluoromethyl, cyano, azido, heterocyclyl,alkylaryl, or an aromatic or heteroaromatic moiety.

Unless the number of carbons is otherwise specified, “lower alkyl” asused herein means an alkyl group, as defined above, but having from oneto five carbon atoms in its backbone structure. “Lower alkenyl” and“lower alkynyl” have chain lengths of, for example, 2-5 carbon atoms.

The term “acyl” includes compounds and moieties which contain the acylradical (CH₃CO—) or a carbonyl group. It includes substituted acylmoieties. The term “substituted acyl” includes acyl groups where one ormore of the hydrogen atoms are replaced by for example, alkyl groups,alkynyl groups, halogens, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy,alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl,arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl,dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate,phosphonato, phosphinato, cyano, amino (including alkyl amino,dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino(including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido),amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate,sulfates, alkylsulfinyl, sulfonato, sulfamoyl, sulfonamido, nitro,trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an aromaticor heteroaromatic moiety.

The term “acylamino” includes moieties wherein an acyl moiety is bondedto an amino group. For example, the term includes alkylcarbonylamino,arylcarbonylamino, carbamoyl and ureido groups.

The term “aroyl” includes compounds and moieties with an aryl orheteroaromatic moiety bound to a carbonyl group. Examples of aroylgroups include phenylcarboxy, naphthyl carboxy, etc.

The terms “alkoxyalkyl”, “alkylaminoalkyl” and “thioalkoxyalkyl” includealkyl groups, as described above, which further include oxygen, nitrogenor sulfur atoms replacing one or more carbons of the hydrocarbonbackbone, e.g., oxygen, nitrogen or sulfur atoms.

The term “alkoxy” includes substituted and unsubstituted alkyl, alkenyl,and alkynyl groups covalently linked to an oxygen atom. Examples ofalkoxy groups include methoxy, ethoxy, isopropyloxy, propoxy, butoxy,and pentoxy groups. Examples of substituted alkoxy groups includehalogenated alkoxy groups. The alkoxy groups can be substituted withgroups such as alkenyl, alkynyl, halogen, hydroxyl, alkylcarbonyloxy,arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate,alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aminocarbonyl,alkylaminocarbonyl, dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl,phosphate, phosphonato, phosphinato, cyano, amino (including alkylamino, dialkylamino, arylamino, diarylamino, and alkylarylamino),acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyland ureido), amidino, imino, sulfhydryl, alkylthio, arylthio,thiocarboxylate, sulfates, alkylsulfinyl, sulfonato, sulfamoyl,sulfonamido, nitro, trifluoromethyl, cyano, azido, heterocyclyl,alkylaryl, or an aromatic or heteroaromatic moieties. Examples ofhalogen substituted alkoxy groups include, but are not limited to,fluoromethoxy, difluoromethoxy, trifluoromethoxy, chloromethoxy,dichloromethoxy, trichloromethoxy, etc.

The term “amine” or “amino” includes compounds where a nitrogen atom iscovalently bonded to at least one carbon or heteroatom. The term “alkylamino” includes groups and compounds wherein the nitrogen is bound to atleast one additional alkyl group. The term “dialkyl amino” includesgroups wherein the nitrogen atom is bound to at least two additionalalkyl groups. The term “arylamino” and “diarylamino” include groupswherein the nitrogen is bound to at least one or two aryl groups,respectively. The term “alkylarylamino,” “alkylaminoaryl” or“arylaminoalkyl” refers to an amino group which is bound to at least onealkyl group and at least one aryl group. The term “alkaminoalkyl” refersto an alkyl, alkenyl, or alkynyl group bound to a nitrogen atom which isalso bound to an alkyl group.

The term “amide” or “aminocarbonyl” includes compounds or moieties whichcontain a nitrogen atom which is bound to the carbon of a carbonyl or athiocarbonyl group. The term includes “alkaminocarbonyl” or“alkylaminocarbonyl” groups which include alkyl, alkenyl, aryl oralkynyl groups bound to an amino group bound to a carbonyl group. Itincludes arylaminocarbonyl groups which include aryl or heteroarylmoieties bound to an amino group which is bound to the carbon of acarbonyl or thiocarbonyl group. The terms “alkylaminocarbonyl,”“alkenylaminocarbonyl,” “alkynylaminocarbonyl,” “arylaminocarbonyl,”“alkylcarbonylamino,” “alkenylcarbonylamino,” “alkynylcarbonylamino,”and “arylcarbonylamino” are included in term “amide.” Amides alsoinclude urea groups (aminocarbonylamino) and carbamates(oxycarbonylamino).

The term “carbonyl” or “carboxy” includes compounds and moieties whichcontain a carbon connected with a double bond to an oxygen atom. Thecarbonyl can be further substituted with any moiety which allows thecompounds of the invention to perform its intended function. Forexample, carbonyl moieties may be substituted with alkyls, alkenyls,alkynyls, aryls, alkoxy, aminos, etc. Examples of moieties which containa carbonyl include aldehydes, ketones, carboxylic acids, amides, esters,anhydrides, etc.

The term “thiocarbonyl” or “thiocarboxy” includes compounds and moietieswhich contain a carbon connected with a double bond to a sulfur atom.

The term “ether” includes compounds or moieties which contain an oxygenbonded to two different carbon atoms or heteroatoms. For example, theterm includes “alkoxyalkyl” which refers to an alkyl, alkenyl, oralkynyl group covalently bonded to an oxygen atom which is covalentlybonded to another alkyl group.

The term “ester” includes compounds and moieties which contain a carbonor a heteroatom bound to an oxygen atom which is bonded to the carbon ofa carbonyl group. The term “ester” includes alkoxycarboxy groups such asmethoxycarbonyl, ethoxycarbonyl, propoxycarbonyl, butoxycarbonyl,pentoxycarbonyl, etc. The alkyl, alkenyl, or alkynyl groups are asdefined above.

The term “thioether” includes compounds and moieties which contain asulfur atom bonded to two different carbon or hetero atoms. Examples ofthioethers include, but are not limited to alkthioalkyls,alkthioalkenyls, and alkthioalkynyls. The term “alkthioalkyls” includecompounds with an alkyl, alkenyl, or alkynyl group bonded to a sulfuratom which is bonded to an alkyl group. Similarly, the term“alkthioalkenyls” and alkthioalkynyls” refer to compounds or moietieswherein an alkyl, alkenyl, or alkynyl group is bonded to a sulfur atomwhich is covalently bonded to an alkynyl group.

The term “hydroxy” or “hydroxyl” includes groups with an —OH or —O⁻.

The term “halogen” includes fluorine, bromine, chlorine, iodine, etc.The term “perhalogenated” generally refers to a moiety wherein allhydrogens are replaced by halogen atoms.

The terms “polycyclyl” or “polycyclic radical” refer to two or morecyclic rings (e.g., cycloalkyls, cycloalkenyls, cycloalkynyls, arylsand/or heterocyclyls) in which two or more carbons are common to twoadjoining rings, e.g., the rings are “fused rings”. Rings that arejoined through non-adjacent atoms are termed “bridged” rings. Each ofthe rings of the polycycle can be substituted with such substituents asdescribed above, as for example, halogen, hydroxyl, alkylcarbonyloxy,arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate,alkylcarbonyl, alkoxycarbonyl, alkylaminoacarbonyl,arylalkylaminocarbonyl, alkenylaminocarbonyl, alkylcarbonyl,arylcarbonyl, arylalkyl carbonyl, alkenylcarbonyl, aminocarbonyl,alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phosphinato, cyano,amino (including alkyl amino, dialkylamino, arylamino, diarylamino, andalkylarylamino), acylamino (including alkylcarbonylamino,arylcarbonylamino, carbamoyl and ureido), amidino, imino, sulfhydryl,alkylthio, arylthio, thiocarboxylate, sulfates, alkylsulfinyl,sulfonato, sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido,heterocyclyl, alkyl, alkylaryl, or an aromatic or heteroaromatic moiety.

The term “heteroatom” includes atoms of any element other than carbon orhydrogen. Preferred heteroatoms are nitrogen, oxygen, sulfur andphosphorus.

The term “prodrug moiety” includes moieties which can be metabolized invivo to a hydroxyl group and moieties which may advantageously remainesterified in vivo. Preferably, the prodrugs moieties are metabolized invivo by esterases or by other mechanisms to hydroxyl groups or otheradvantageous groups. Examples of prodrugs and their uses are well knownin the art (See, e.g., Berge et al. (1977) “Pharmaceutical Salts”, J.Pharm. Sci. 66:1-19). The prodrugs can be prepared in situ during thefinal isolation and purification of the compounds, or by separatelyreacting the purified compound in its free acid form or hydroxyl with asuitable esterifying agent. Hydroxyl groups can be converted into estersvia treatment with a carboxylic acid. Examples of prodrug moietiesinclude substituted and unsubstituted, branch or unbranched lower alkylester moieties, (e.g., propionoic acid esters), lower alkenyl esters,di-lower alkyl-amino lower-alkyl esters (e.g., dimethylaminoethylester), acylamino lower alkyl esters (e.g., acetyloxymethyl ester),acyloxy lower alkyl esters (e.g., pivaloyloxymethyl ester), aryl esters(phenyl ester), aryl-lower alkyl esters (e.g., benzyl ester),substituted (e.g., with methyl, halo, or methoxy substituents) aryl andaryl-lower alkyl esters, amides, lower-alkyl amides, di-lower alkylamides, and hydroxy amides. Preferred prodrug moieties are propionoicacid esters and acyl esters.

It will be noted that the structure of some of the tetracyclinecompounds of this invention includes asymmetric carbon atoms. It is tobe understood accordingly that the isomers arising from such asymmetry(e.g., all enantiomers and diastereomers) are included within the scopeof this invention, unless indicated otherwise. Such isomers can beobtained in substantially pure form by classical separation techniquesand by stereochemically controlled synthesis. Furthermore, thestructures and other compounds and moieties discussed in thisapplication also include all tautomers thereof.

The invention also pertains to methods for treating a tetracyclineresponsive states in subjects, by administering to a subject aneffective amount of a tetracycline compound of the invention (e.g., acompound of Formula (I)), such that the tetracycline responsive state istreated.

The term “treating” includes curing as well as ameliorating at least onesymptom of the state, disease or disorder, e.g., the tetracyclinecompound responsive state.

The language “tetracycline compound responsive state” or “tetracyclineresponsive state” includes states which can be treated, prevented, orotherwise ameliorated by the administration of a tetracycline compoundof the invention, e.g., a 3, 10, and/or 12a substituted tetracyclinecompound. Tetracycline compound responsive states include bacterial,viral, and fungal infections (including those which are resistant toother tetracycline compounds), cancer (e.g., prostate, breast, colon,lung melanoma and lymph cancers and other disorders characterized byunwanted cellular proliferation, including, but not limited to, thosedescribed in U.S. Pat. No. 6,100,248), arthritis, osteoporosis,diabetes, and other states for which tetracycline compounds have beenfound to be active (see, for example, U.S. Pat. Nos. 5,789,395;5,834,450; 6,277,061 and 5,532,227, each of which is expresslyincorporated herein by reference). Compounds of the invention can beused to prevent or control important mammalian and veterinary diseasessuch as diarrhea, urinary tract infections, infections of skin and skinstructure, ear, nose and throat infections, wound infection, mastitisand the like. In addition, methods for treating neoplasms usingtetracycline compounds of the invention are also included (van derBozert et al., Cancer Res., 48:6686-6690 (1988)). In a furtherembodiment, the tetracycline responsive state is not a bacterialinfection. In another embodiment, the tetracycline compounds of theinvention are essentially non-antibacterial. For example,non-antibacterial tetracycline compounds of the invention may have MICvalues greater than about 4 μg/ml (as measured by assays known in theart and/or the assay given in Example 2).

Tetracycline compound responsive states also include inflammatoryprocess associated states (IPAS). The term “inflammatory processassociated state” includes states in which inflammation or inflammatoryfactors (e.g., matrix metalloproteinases (MMPs), nitric oxide (NO), TNF,interleukins, plasma proteins, cellular defense systems, cytokines,lipid metabolites, proteases, toxic radicals, adhesion molecules, etc.)are involved or are present in an area in aberrant amounts, e.g., inamounts which may be advantageous to alter, e.g., to benefit thesubject. The inflammatory process is the response of living tissue todamage. The cause of inflammation may be due to physical damage,chemical substances, micro-organisms, tissue necrosis, cancer or otheragents. Acute inflammation is short-lasting, lasting only a few days. Ifit is longer lasting however, then it may be referred to as chronicinflammation.

IPAF's include inflammatory disorders. Inflammatory disorders aregenerally characterized by heat, redness, swelling, pain and loss offunction. Examples of causes of inflammatory disorders include, but arenot limited to, microbial infections (e.g., bacterial and fungalinfections), physical agents (e.g., burns, radiation, and trauma),chemical agents (e.g., toxins and caustic substances), tissue necrosisand various types of immunologic reactions.

Examples of inflammatory disorders include, but are not limited to,osteoarthritis, rheumatoid arthritis, acute and chronic infections(bacterial and fungal, including diphtheria and pertussis); acute andchronic bronchitis, sinusitis, and upper respiratory infections,including the common cold; acute and chronic gastroenteritis andcolitis; acute and chronic cystitis and urethritis; acute and chronicdermatitis; acute and chronic conjunctivitis; acute and chronicserositis (pericarditis, peritonitis, synovitis, pleuritis andtendinitis); uremic pericarditis; acute and chronic cholecystis; acuteand chronic vaginitis; acute and chronic uveitis; drug reactions; insectbites; burns (thermal, chemical, and electrical); and sunburn.

Tetracycline compound responsive states also include NO associatedstates. The term “NO associated state” includes states which involve orare associated with nitric oxide (NO) or inducible nitric oxide synthase(iNOS). NO associated state includes states which are characterized byaberrant amounts of NO and/or iNOS. Preferably, the NO associated statecan be treated by administering tetracycline compounds of the invention,e.g., a 3, 10, and/or 12a substituted tetracycline compound. Thedisorders, diseases and states described in U.S. Pat. Nos. 6,231,894;6,015,804; 5,919,774; and 5,789,395 are also included as NO associatedstates. The entire contents of each of these patents are herebyincorporated herein by reference.

Other examples of NO associated states include, but are not limited to,malaria, senescence, diabetes, vascular stroke, neurodegenerativedisorders (Alzheimer's disease & Huntington's disease), cardiac disease(reperfusion-associated injury following infarction), juvenile diabetes,inflammatory disorders, osteoarthritis, rheumatoid arthritis, acute,recurrent and chronic infections (bacterial, viral and fungal); acuteand chronic bronchitis, sinusitis, and respiratory infections, includingthe common cold; acute and chronic gastroenteritis and colitis; acuteand chronic cystitis and urethritis; acute and chronic dermatitis; acuteand chronic conjunctivitis; acute and chronic serositis (pericarditis,peritonitis, synovitis, pleuritis and tendonitis); uremic pericarditis;acute and chronic cholecystis; cystic fibrosis, acute and chronicvaginitis; acute and chronic uveitis; drug reactions; insect bites;burns (thermal, chemical, and electrical); and sunburn.

The term “inflammatory process associated state” also includes, in oneembodiment, matrix metalloproteinase associated states (MMPAS). MMPASinclude states characterized by aberrant amounts of MMPs or MMPactivity. These are also include as tetracycline compound responsivestates which may be treated using compounds of the invention, e.g., 3,10, and/or 12a substituted tetracycline compounds.

Examples of matrix metalloproteinase associated states (“MMPAS's”)include, but are not limited to, arteriosclerosis, corneal ulceration,emphysema, osteoarthritis, multiple sclerosis (Liedtke et al., Ann.Neurol. 1998, 44:35-46; Chandler et al., J. Neuroimmunol. 1997,72:155-71), osteosarcoma, osteomyelitis, bronchiectasis, chronicpulmonary obstructive disease, skin and eye diseases, periodontitis,osteoporosis, rheumatoid arthritis, ulcerative colitis, inflammatorydisorders, tumor growth and invasion (Stetler-Stevenson et al., Annu.Rev. Cell Biol. 1993, 9:541-73; Tryggvason et al., Biochim. Biophys.Acta 1987, 907:191-217; Li et al., Mol. Carcinog. 1998, 22:84-89)),metastasis, acute lung injury, stroke, ischemia, diabetes, aortic orvascular aneurysms, skin tissue wounds, dry eye, bone and cartilagedegradation (Greenwald et al., Bone 1998, 22:33-38; Ryan et al., Curr.Op. Rheumatol. 1996, 8; 238-247). Other MMPAS include those described inU.S. Pat. Nos. 5,459,135; 5,321,017; 5,308,839; 5,258,371; 4,935,412;4,704,383, 4,666,897, and RE 34,656, incorporated herein by reference intheir entirety.

In another embodiment, the tetracycline compound responsive state iscancer. Examples of cancers which the tetracycline compounds of theinvention may be useful to treat include all solid tumors, i.e.,carcinomas e.g., adenocarcinomas, and sarcomas. Adenocarcinomas arecarcinomas derived from glandular tissue or in which the tumor cellsform recognizable glandular structures. Sarcomas broadly include tumorswhose cells are embedded in a fibrillar or homogeneous substance likeembryonic connective tissue. Examples of carcinomas which may be treatedusing the methods of the invention include, but are not limited to,carcinomas of the prostate, breast, ovary, testis, lung, colon, andbreast. The methods of the invention are not limited to the treatment ofthese tumor types, but extend to any solid tumor derived from any organsystem. Examples of treatable cancers include, but are not limited to,colon cancer, bladder cancer, breast cancer, melanoma, ovariancarcinoma, prostatic carcinoma, lung cancer, and a variety of othercancers as well. The methods of the invention also cause the inhibitionof cancer growth in adenocarcinomas, such as, for example, those of theprostate, breast, kidney, ovary, testes, and colon.

In an embodiment, the tetracycline responsive state of the invention iscancer. The invention pertains to a method for treating a subjectsuffering or at risk of suffering from cancer, by administering aneffective amount of a substituted tetracycline compound, such thatinhibition cancer cell growth occurs, i.e., cellular proliferation,invasiveness, metastasis, or tumor incidence is decreased, slowed, orstopped. The inhibition may result from inhibition of an inflammatoryprocess, down-regulation of an inflammatory process, some othermechanism, or a combination of mechanisms. Alternatively, thetetracycline compounds may be useful for preventing cancer recurrence,for example, to treat residual cancer following surgical resection orradiation therapy. The tetracycline compounds useful according to theinvention are especially advantageous as they are substantiallynon-toxic compared to other cancer treatments. In a further embodiment,the compounds of the invention are administered in combination withstandard cancer therapy, such as, but not limited to, chemotherapy.

Examples of tetracycline responsive states also include neurologicaldisorders which include both neuropsychiatric and neurodegenerativedisorders, but are not limited to, such as Alzheimer's disease,dementias related to Alzheimer's disease (such as Pick's disease),Parkinson's and other Lewy diffuse body diseases, senile dementia,Huntington's disease, Gilles de la Tourette's syndrome, multiplesclerosis, amylotrophic lateral sclerosis (ALS), progressivesupranuclear palsy, epilepsy, and Creutzfeldt-Jakob disease; autonomicfunction disorders such as hypertension and sleep disorders, andneuropsychiatric disorders, such as depression, schizophrenia,schizoaffective disorder, Korsakoff's psychosis, mania, anxietydisorders, or phobic disorders; learning or memory disorders, e.g.,amnesia or age-related memory loss, attention deficit disorder,dysthymic disorder, major depressive disorder, mania,obsessive-compulsive disorder, psychoactive substance use disorders,anxiety, phobias, panic disorder, as well as bipolar affective disorder,e.g., severe bipolar affective (mood) disorder (BP-1), bipolar affectiveneurological disorders, e.g., migraine and obesity. Further neurologicaldisorders include, for example, those listed in the American PsychiatricAssociation's Diagnostic and Statistical manual of Mental Disorders(DSM), the most current version of which is incorporated herein byreference in its entirety.

The language “in combination with” another therapeutic agent ortreatment includes co-administration of the tetracycline compound,(e.g., inhibitor) and with the other therapeutic agent or treatment,administration of the tetracycline compound first, followed by the othertherapeutic agent or treatment and administration of the othertherapeutic agent or treatment first, followed by the tetracyclinecompound. The other therapeutic agent may be any agent who is known inthe art to treat, prevent, or reduce the symptoms of an IPAS.Furthermore, the other therapeutic agent may be any agent of benefit tothe patient when administered in combination with the administration ofan tetracycline compound. In one embodiment, the cancers treated bymethods of the invention include those described in U.S. Pat. Nos.6,100,248; 5,843,925; 5,837,696; or 5,668,122, incorporated herein byreference in their entirety.

In another embodiment, the tetracycline compound responsive state isdiabetes, e.g., juvenile diabetes, diabetes mellitus, diabetes type I,or diabetes type II. In a further embodiment, protein glycosylation isnot affected by the administration of the tetracycline compounds of theinvention. In another embodiment, the tetracycline compound of theinvention is administered in combination with standard diabetictherapies, such as, but not limited to insulin therapy. In a furtherembodiment, the IPAS includes disorders described in U.S. Pat. Nos.5,929,055; and 5,532,227, incorporated herein by reference in theirentirety.

In another embodiment, the tetracycline compound responsive state is abone mass disorder. Bone mass disorders include disorders where asubjects bones are disorders and states where the formation, repair orremodeling of bone is advantageous. For examples bone mass disordersinclude osteoporosis (e.g., a decrease in bone strength and density),bone fractures, bone formation associated with surgical procedures(e.g., facial reconstruction), osteogenesis imperfecta (brittle bonedisease), hypophosphatasia, Paget's disease, fibrous dysplasia,osteopetrosis, myeloma bone disease, and the depletion of calcium inbone, such as that which is related to primary hyperparathyroidism. Bonemass disorders include all states in which the formation, repair orremodeling of bone is advantageous to the subject as well as all otherdisorders associated with the bones or skeletal system of a subjectwhich can be treated with the tetracycline compounds of the invention.In a further embodiment, the bone mass disorders include those describedin U.S. Pat. Nos. 5,459,135; 5,231,017; 5,998,390; 5,770,588; RE 34,656;5,308,839; 4,925,833; 3,304,227; and 4,666,897, each of which is herebyincorporated herein by reference in its entirety.

In another embodiment, the tetracycline compound responsive state isacute lung injury. Acute lung injuries include adult respiratorydistress syndrome (ARDS), post-pump syndrome (PPS), and trauma. Traumaincludes any injury to living tissue caused by an extrinsic agent orevent. Examples of trauma include, but are not limited to, crushinjuries, contact with a hard surface, or cutting or other damage to thelungs.

The invention also pertains to a method for treating acute lung injuryby administering a substituted tetracycline compound of the invention.

The tetracycline responsive states of the invention also include chroniclung disorders. The invention pertains to methods for treating chroniclung disorders by administering a tetracycline compound, such as thosedescribed herein. The method includes administering to a subject aneffective amount of a substituted tetracycline compound such that thechronic lung disorder is treated. Examples of chronic lung disordersinclude, but are not limited, to asthma, cystic fibrosis, and emphysema.In a further embodiment, the tetracycline compounds of the inventionused to treat acute and/or chronic lung disorders such as thosedescribed in U.S. Pat. Nos. 5,977,091; 6,043,231; 5,523,297; and5,773,430, each of which is hereby incorporated herein by reference inits entirety.

In yet another embodiment, the tetracycline compound responsive state isischemia, stroke, or ischemic stroke. The invention also pertains to amethod for treating ischemia, stroke, or ischemic stroke byadministering an effective amount of a substituted tetracycline compoundof the invention. In a further embodiment, the tetracycline compounds ofthe invention are used to treat such disorders as described in U.S. Pat.Nos. 6,231,894; 5,773,430; 5,919,775 or 5,789,395, incorporated hereinby reference.

In another embodiment, the tetracycline compound responsive state is askin wound. The invention also pertains, at least in part, to a methodfor improving the healing response of the epithelialized tissue (e.g.,skin, mucusae) to acute traumatic injury (e.g., cut, burn, scrape,etc.). The method may include using a tetracycline compound of theinvention (which may or may not have antibacterial activity) to improvethe capacity of the epithelialized tissue to heal acute wounds. Themethod may increase the rate of collagen accumulation of the healingtissue. The method may also decrease the proteolytic activity in theepthithelialized tissue by decreasing the collagenolytic and/orgellatinolytic activity of MMPs. In a further embodiment, thetetracycline compound of the invention is administered to the surface ofthe skin (e.g., topically). In a further embodiment, the tetracyclinecompound of the invention used to treat a skin wound, and other suchdisorders as described in, for example, U.S. Pat. Nos. 5,827,840;4,704,383; 4,935,412; 5,258,371; 5,308,8391 5,459,135; 5,532,227; and6,015,804; each of which is incorporated herein by reference in itsentirety.

In yet another embodiment, the tetracycline compound responsive state isan aortic or vascular aneurysm in vascular tissue of a subject (e.g., asubject having or at risk of having an aortic or vascular aneurysm,etc.). The tetracycline compound may by effective to reduce the size ofthe vascular aneurysm or it may be administered to the subject prior tothe onset of the vascular aneurysm such that the aneurysm is prevented.In one embodiment, the vascular tissue is an artery, e.g., the aorta,e.g., the abdominal aorta. In a further embodiment, the tetracyclinecompounds of the invention are used to treat disorders described in U.S.Pat. Nos. 6,043,225 and 5,834,449, incorporated herein by reference intheir entirety.

Bacterial infections may be caused by a wide variety of gram positiveand gram negative bacteria. The compounds of the invention are useful asantibiotics against organisms which are resistant to other tetracyclinecompounds. The antibiotic activity of the tetracycline compounds of theinvention may be determined using the method discussed in Example 2, orby using the in vitro standard broth dilution method described in Waitz,J. A., National Commission for Clinical Laboratory Standards, DocumentM7-A2, vol. 10, no. 8, pp. 13-20, 2^(nd) edition, Villanova, Pa. (1990).

The tetracycline compounds may also be used to treat infectionstraditionally treated with tetracycline compounds such as, for example,rickettsiae; a number of gram-positive and gram-negative bacteria; andthe agents responsible for lymphogranuloma venereum, inclusionconjunctivitis, psittacosis. The tetracycline compounds may be used totreat infections of, e.g., K. pneumoniae, Salmonella, E. hirae, A.baumanii, B. catarrhalis, H. influenzae, P. aeruginosa, E. faecium, E.coli, S. aureus or E. faecalis. In one embodiment, the tetracyclinecompound is used to treat a bacterial infection that is resistant toother tetracycline antibiotic compounds. The tetracycline compound ofthe invention may be administered with a pharmaceutically acceptablecarrier.

The language “effective amount” of the compound is that amount necessaryor sufficient to treat or prevent a tetracycline compound responsivestate. The effective amount can vary depending on such factors as thesize and weight of the subject, the type of illness, or the particulartetracycline compound. For example, the choice of the tetracyclinecompound can affect what constitutes an “effective amount”. One ofordinary skill in the art would be able to study the aforementionedfactors and make the determination regarding the effective amount of thetetracycline compound without undue experimentation.

The invention also pertains to methods of treatment againstmicroorganism infections and associated diseases. The methods includeadministration of an effective amount of one or more tetracyclinecompounds to a subject. The subject can be either a plant or,advantageously, an animal, e.g., a mammal, e.g., a human.

In the therapeutic methods of the invention, one or more tetracyclinecompounds of the invention may be administered alone to a subject, ormore typically a compound of the invention will be administered as partof a pharmaceutical composition in mixture with conventional excipient,i.e., pharmaceutically acceptable organic or inorganic carriersubstances suitable for parenteral, oral or other desired administrationand which do not deleteriously react with the active compounds and arenot deleterious to the recipient thereof.

The invention also pertains to pharmaceutical compositions comprising atherapeutically effective amount of a tetracycline compound (e.g., a 3,10, and/or 12a substituted tetracycline compound) and, optionally, apharmaceutically acceptable carrier.

The language “pharmaceutically acceptable carrier” includes substancescapable of being coadministered with the tetracycline compound(s), andwhich allow both to perform their intended function, e.g., treat orprevent a tetracycline responsive state. Suitable pharmaceuticallyacceptable carriers include but are not limited to water, saltsolutions, alcohol, vegetable oils, polyethylene glycols, gelatin,lactose, amylose, magnesium stearate, talc, silicic acid, viscousparaffin, perfume oil, fatty acid monoglycerides and diglycerides,petroethral fatty acid esters, hydroxymethyl-cellulose,polyvinylpyrrolidone, etc. The pharmaceutical preparations can besterilized and if desired mixed with auxiliary agents, e.g., lubricants,preservatives, stabilizers, wetting agents, emulsifiers, salts forinfluencing osmotic pressure, buffers, colorings, flavorings and/oraromatic substances and the like which do not deleteriously react withthe active compounds of the invention.

The tetracycline compounds of the invention that are basic in nature arecapable of forming a wide variety of salts with various inorganic andorganic acids. The acids that may be used to prepare pharmaceuticallyacceptable acid addition salts of the tetracycline compounds of theinvention that are basic in nature are those that form non-toxic acidaddition salts, i.e., salts containing pharmaceutically acceptableanions, such as the hydrochloride, hydrobromide, hydroiodide, nitrate,sulfate, bisulfate, phosphate, acid phosphate, isonicotinate, acetate,lactate, salicylate, citrate, acid citrate, tartrate, pantothenate,bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate,gluconate, glucaronate, saccharate, formate, benzoate, glutamate,methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonateand palmoate [i.e., 1,1′-methylene-bis-(2-hydroxy-3-naphthoate)] salts.Although such salts must be pharmaceutically acceptable foradministration to a subject, e.g., a mammal, it is often desirable inpractice to initially isolate a tetracycline compound of the inventionfrom the reaction mixture as a pharmaceutically unacceptable salt andthen simply convert the latter back to the free base compound bytreatment with an alkaline reagent and subsequently convert the latterfree base to a pharmaceutically acceptable acid addition salt. The acidaddition salts of the base compounds of this invention are readilyprepared by treating the base compound with a substantially equivalentamount of the chosen mineral or organic acid in an aqueous solventmedium or in a suitable organic solvent, such as methanol or ethanol.Upon careful evaporation of the solvent, the desired solid salt isreadily obtained. The preparation of other tetracycline compounds of theinvention not specifically described in the foregoing experimentalsection can be accomplished using combinations of the reactionsdescribed above that will be apparent to those skilled in the art.

The preparation of other tetracycline compounds of the invention notspecifically described in the foregoing experimental section can beaccomplished using combinations of the reactions described above thatwill be apparent to those skilled in the art.

The tetracycline compounds of the invention that are acidic in natureare capable of forming a wide variety of base salts. The chemical basesthat may be used as reagents to prepare pharmaceutically acceptable basesalts of those tetracycline compounds of the invention that are acidicin nature are those that form non-toxic base salts with such compounds.Such non-toxic base salts include, but are not limited to those derivedfrom such pharmaceutically acceptable cations such as alkali metalcations (e.g., potassium and sodium) and alkaline earth metal cations(e.g., calcium and magnesium), ammonium or water-soluble amine additionsalts such as N-methylglucamine-(meglumine), and the loweralkanolammonium and other base salts of pharmaceutically acceptableorganic amines. The pharmaceutically acceptable base addition salts oftetracycline compounds of the invention that are acidic in nature may beformed with pharmaceutically acceptable cations by conventional methods.Thus, these salts may be readily prepared by treating the tetracyclinecompound of the invention with an aqueous solution of the desiredpharmaceutically acceptable cation and evaporating the resultingsolution to dryness, preferably under reduced pressure. Alternatively, alower alkyl alcohol solution of the tetracycline compound of theinvention may be mixed with an alkoxide of the desired metal and thesolution subsequently evaporated to dryness.

The preparation of other tetracycline compounds of the invention notspecifically described in the foregoing experimental section can beaccomplished using combinations of the reactions described above thatwill be apparent to those skilled in the art.

The tetracycline compounds of the invention and pharmaceuticallyacceptable salts thereof can be administered via either the oral,parenteral or topical routes. In general, these compounds are mostdesirably administered in effective dosages, depending upon the weightand condition of the subject being treated and the particular route ofadministration chosen. Variations may occur depending upon the speciesof the subject being treated and its individual response to saidmedicament, as well as on the type of pharmaceutical formulation chosenand the time period and interval at which such administration is carriedout.

The pharmaceutical compositions of the invention may be administeredalone or in combination with other known compositions for treatingtetracycline responsive states in a subject, e.g., a mammal. Preferredmammals include pets (e.g., cats, dogs, ferrets, etc.), farm animals(cows, sheep, pigs, horses, goats, etc.), lab animals (rats, mice,monkeys, etc.), and primates (chimpanzees, humans, gorillas). Thelanguage “in combination with” a known composition is intended toinclude simultaneous administration of the composition of the inventionand the known composition, administration of the composition of theinvention first, followed by the known composition and administration ofthe known composition first, followed by the composition of theinvention. Any of the therapeutically composition known in the art fortreating tetracycline responsive states can be used in the methods ofthe invention.

The tetracycline compounds of the invention may be administered alone orin combination with pharmaceutically acceptable carriers or diluents byany of the routes previously mentioned, and the administration may becarried out in single or multiple doses. For example, the noveltherapeutic agents of this invention can be administered advantageouslyin a wide variety of different dosage forms, i.e., they may be combinedwith various pharmaceutically acceptable inert carriers in the form oftablets, capsules, lozenges, troches, hard candies, powders, sprays,creams, salves, suppositories, jellies, gels, pastes, lotions,ointments, aqueous suspensions, injectable solutions, elixirs, syrups,and the like. Such carriers include solid diluents or fillers, sterileaqueous media and various non-toxic organic solvents, etc. Moreover,oral pharmaceutical compositions can be suitably sweetened and/orflavored. In general, the therapeutically-effective compounds of thisinvention are present in such dosage forms at concentration levelsranging from about 5.0% to about 70% by weight.

For oral administration, tablets containing various excipients such asmicrocrystalline cellulose, sodium citrate, calcium carbonate, dicalciumphosphate and glycine may be employed along with various disintegrantssuch as starch (and preferably corn, potato or tapioca starch), alginicacid and certain complex silicates, together with granulation binderslike polyvinylpyrrolidone, sucrose, gelatin and acacia. Additionally,lubricating agents such as magnesium stearate, sodium lauryl sulfate andtalc are often very useful for tabletting purposes. Solid compositionsof a similar type may also be employed as fillers in gelatin capsules;preferred materials in this connection also include lactose or milksugar as well as high molecular weight polyethylene glycols. Whenaqueous suspensions and/or elixirs are desired for oral administration,the active ingredient may be combined with various sweetening orflavoring agents, coloring matter or dyes, and, if so desired,emulsifying and/or suspending agents as well, together with suchdiluents as water, ethanol, propylene glycol, glycerin and various likecombinations thereof.

For parenteral administration (including intraperitoneal, subcutaneous,intravenous, intradermal or intramuscular injection), solutions of atherapeutic compound of the present invention in either sesame or peanutoil or in aqueous propylene glycol may be employed. The aqueoussolutions should be suitably buffered (preferably pH greater than 8) ifnecessary and the liquid diluent first rendered isotonic. These aqueoussolutions are suitable for intravenous injection purposes. The oilysolutions are suitable for intraarticular, intramuscular andsubcutaneous injection purposes. The preparation of all these solutionsunder sterile conditions is readily accomplished by standardpharmaceutical techniques well known to those skilled in the art. Forparenteral application, examples of suitable preparations includesolutions, preferably oily or aqueous solutions as well as suspensions,emulsions, or implants, including suppositories. Therapeutic compoundsmay be formulated in sterile form in multiple or single dose formatssuch as being dispersed in a fluid carrier such as sterile physiologicalsaline or 5% saline dextrose solutions commonly used with injectables.

Additionally, it is also possible to administer the compounds of thepresent invention topically when treating inflammatory conditions of theskin. Examples of methods of topical administration include transdermal,buccal or sublingual application. For topical applications, therapeuticcompounds can be suitably admixed in a pharmacologically inert topicalcarrier such as a gel, an ointment, a lotion or a cream. Such topicalcarriers include water, glycerol, alcohol, propylene glycol, fattyalcohols, triglycerides, fatty acid esters, or mineral oils. Otherpossible topical carriers are liquid petrolatum, isopropylpalmitate,polyethylene glycol, ethanol 95%, polyoxyethylene monolauriate 5% inwater, sodium lauryl sulfate 5% in water, and the like. In addition,materials such as anti-oxidants, humectants, viscosity stabilizers andthe like also may be added if desired.

For enteral application, particularly suitable are tablets, dragees orcapsules having talc and/or carbohydrate carrier binder or the like, thecarrier preferably being lactose and/or corn starch and/or potatostarch. A syrup, elixir or the like can be used wherein a sweetenedvehicle is employed. Sustained release compositions can be formulatedincluding those wherein the active component is derivatized withdifferentially degradable coatings, e.g., by microencapsulation,multiple coatings, etc.

In addition to treatment of human subjects, the therapeutic methods ofthe invention also will have significant veterinary applications, e.g.for treatment of livestock such as cattle, sheep, goats, cows, swine andthe like; poultry such as chickens, ducks, geese, turkeys and the like;horses; and pets such as dogs and cats. Also, the compounds of theinvention may be used to treat non-animal subjects, such as plants.

It will be appreciated that the actual preferred amounts of activecompounds used in a given therapy will vary according to the specificcompound being utilized, the particular compositions formulated, themode of application, the particular site of administration, etc. Optimaladministration rates for a given protocol of administration can bereadily ascertained by those skilled in the art using conventionaldosage determination tests conducted with regard to the foregoingguidelines.

In general, compounds of the invention for treatment can be administeredto a subject in dosages used in prior tetracycline therapies. See, forexample, the Physicians' Desk Reference. For example, a suitableeffective dose of one or more compounds of the invention will be in therange of from 0.01 to 100 milligrams per kilogram of body weight ofrecipient per day, preferably in the range of from 0.1 to 50 milligramsper kilogram body weight of recipient per day, more preferably in therange of 1 to 20 milligrams per kilogram body weight of recipient perday. The desired dose is suitably administered once daily, or severalsub-doses, e.g. 2 to 5 sub-doses, are administered at appropriateintervals through the day, or other appropriate schedule. It will alsobe understood that normal, conventionally known precautions will betaken regarding the administration of tetracyclines generally to ensuretheir efficacy under normal use circumstances. Especially when employedfor therapeutic treatment of humans and animals in vivo, thepractitioner should take all sensible precautions to avoidconventionally known contradictions and toxic effects. Thus, theconventionally recognized adverse reactions of gastrointestinal distressand inflammations, the renal toxicity, hypersensitivity reactions,changes in blood, and impairment of absorption through aluminum,calcium, and magnesium ions should be duly considered in theconventional manner.

Furthermore, the invention also pertains to the use of a tetracyclinecompound of formula I, for the preparation of a medicament. Themedicament may include a pharmaceutically acceptable carrier and thetetracycline compound is an effective amount, e.g., an effective amountto treat a tetracycline responsive state.

EXEMPLIFICATION OF THE INVENTION

Compounds of the invention may be made as described below and/or byusing literature techniques known to those of ordinary skill in the art.

Example 1 Synthesis of Compounds of the Invention Synthesis of3-Benzyloxysancycline

60% NaH in a mineral oil dispersion (100 mg, 2.5 mmol) was added insmall portions to a stirred solution of sancycline (0.5 g, 1.20 mmol) inDMF (5 mL) at room temperature. The resulting suspension was stirred atroom temperature for 5 minutes. Benzyl bromide (0.143 mL, 1.2 mmol) wasadded and heated at 60° C. for 16 hours. The reaction mixture was thencooled to room temperature and quenched with ether (100 mL). The etherwas decanted and the remaining solid was dissolved in MeOH/water. Theproduct was purified by preparative HPLC and converted to the HCl salt,yielding 3-benzyloxysancycline as a light yellow solid.

Synthesis of 3,10-Dibenzyloxysancycline

60% NaH in a mineral oil dispersion (192 mg, 4.8 mmol) was added insmall portions to a stirred solution of sancycline (0.5 g, 1.20 mmol) inDMF (5 mL) at room temperature. The resulting suspension was stirred atroom temperature for 5 minutes. Benzyl bromide (0.43 mL, 3.6 mmol) wasadded and the reaction mixture was heated at 60° C. for 1 hour. Thereaction mixture was subsequently cooled to room temperature andquenched with ether (100 mL). The ether was the removed by decanting andthe remaining solid was dissolved in MeOH/water. The product waspurified by preparative HPLC and converted to the HCl salt to yield3,10-dibenzyloxysancycline as a light yellow solid.

Synthesis of 10-propyloxysancycline

60% NaH in a mineral oil dispersion (192 mg, 4.8 mmol) was added insmall portions to a stirred solution of sancycline (0.5 g, 1.20 mmol) inDMF (5 mL) at room temperature. The resulting suspension was stirred atroom temperature for 5 minutes. Iodopropane (0.35 mL, 3.6 mmol) wasadded and heated at 60° C. for 1 hour. The reaction mixture was thencooled to room temperature and ether (100 mL) was added. The ether wasdecanted. The remaining solid was dissolved in MeOH/water and theproduct was purified by preparative HPLC and converted to the HCl saltto give 10-propyloxysancycline as a light yellow solid.

Synthesis of 10-Butyloxysancycline

60% NaH in a mineral oil dispersion (192 mg, 4.8 mmol) was added insmall portions to a stirred solution of sancycline (0.5 g, 1.20 mmol) inDMF (5 mL) at room temperature. The resulting suspension was stirred atroom temperature for 5 minutes. Iodobutane (0.411 mL, 3.6 mmol) wasadded and heated at 60° C. for 1 hour. The reaction mixture wassubsequently cooled to room temperature and quenched with ether (100mL). The ether was then decanted and the remaining solid was dissolvedin MeOH/water. The product was purified by preparative HPLC andconverted to the HCl salt to give 10-butyloxysancycline as a lightyellow solid.

Synthesis of 1-Butyloxyminocycline

60% NaH in a mineral oil dispersion (152 mg, 3.8 mmol) was added insmall portions to a stirred solution of minocycline HCl salt (0.5 g,0.95 mmol) in DMF (5 mL) at room temperature. The resulting suspensionwas stirred at room temperature for 5 minutes. Iodobutane (0.325 mL,2.85 mmol) was added and heated at 60° C. for 1 hour. The reactionmixture was cooled to room temperature and quenched with ether (100 mL).The ether was subsequently decanted and the remaining solid wasdissolved in MeOH/water. The product was purified by preparative HPLCand converted to the HCl salt to give 10-butyloxyminocycline as an olivegreen solid.

Synthesis of 3,10-Dibenzyloxyminocycline

60% NaH in a mineral oil dispersion (152 mg, 3.8 mmol) was added insmall portions to a stirred solution of minocycline HCl salt (0.5 g,0.95 mmol) in DMF (5 mL) at room temperature. The resulting suspensionwas stirred at room temperature for 5 minutes. Benzyl bromide (0.34 mL,2.84 mmol) was added and heated at 60° C. for 1 hour. The reactionmixture was then cooled to room temperature and quenched with ether (100mL). The ether was then decanted and the remaining solid was dissolvedin MeOH/water and the product was purified by preparative HPLC. Theproduct was then converted to HCl salt to give3,10-dibenzyloxyminocycline as an olive green solid.

Synthesis of Ethyl-3-(sancyclineoxy)acetate

60% NaH in a mineral oil dispersion (192 mg, 4.8 mmol) was added insmall portions to a stirred solution of sancycline (0.5 g, 1.2 mmol) inDMF (5 mL) at room temperature. The resulting suspension was stirred atroom temperature for 5 minutes. Ethylbromo acetate (0.4 mL, 3.6 mmol)was added and the reaction mixture was heated at 60° C. for 1 hour. Thereaction mixture was cooled to room temperature and quenched with ether(100 mL). The ether was decanted and the remaining solid was dissolvedin MeOH/water. The product was purified by preparative HPLC andconverted to the HCl salt to give ethyl-3-sancyclineoxy) acetate as anolive green solid.

Synthesis of 7-Iodo-10-butyloxysancycline

N-Iodosuccinimide (238 mg, 1.06 mmol) was added in two small portions tostirred solution of crude 10-butyloxysancycline (0.5 g) intrifluoroacetic acid (20 mL) at room temperature. The resulting darkbrown solution was stirred at room temperature for 3 hours. Thetrifluoroacetic acid was evaporated under reduced pressure and theremaining gummy solid was dissolved in MeOH. The product was purified bypreparative HPLC and converted to the HCl salt to give7-iodo-10-butyloxysancycline as a light brown solid.

Synthesis of 10-Butyloxydoxycycline

60% NaH in a mineral oil dispersion (173 mg, 4.3 mmol) was added insmall portions to a stirred solution of doxycycline hydrate (0.5 g, 1.08mmol) in DMF (5 mL) at room temperature. The resulting suspension wasstirred at room temperature for 5 minutes. Iodobutane (0.37 mL, 3.24mmol) was added and the reaction mixture was heated at 60° C. for 1hour. The reaction mixture was then cooled to room temperature andquenched with ether (100 mL). The ether was decanted and the resultinggummy solid was dissolved in MeOH. The product was purified bypreparative HPLC and converted to the HCl salt to give10-butyloxydoxycycline as a light brown solid.

Synthesis of 3,10-Diallyloxysancycline

60% NaH in a mineral oil dispersion (192 mg, 4.8 mmol) was added insmall portions to a stirred solution of sancycline (0.5 g, 1.2 mmol) inDMF (5 mL) at room temperature. The resulting suspension was stirred atroom temperature for 5 minutes. Allyl bromide (0.311 mL, 3.6 mmol) wasadded and heated at 60° C. for 1 hour. The reaction mixture was cooledto room temperature and quenched with ether (100 mL). The ether wasdecanted and the remaining gummy solid was dissolved in MeOH. Theproduct was purified by preparative HPLC and converted to the HCl saltto give 3,10-diallyloxysancycline as a pale brown solid.

Synthesis of 3-Benzyloxy-7-iodosancycline

60% NaH (121 mg, 3.04 mmol) was added in small portions to a stirredsolution of 7-iodosancycline TFA salt (0.5 g, 0.76 mmol) in DMF (10 mL)at room temperature. The resulting suspension was stirred at roomtemperature for 5 minutes. Benzyl bromide (0.277 mL, 2.28 mmol) wasadded and heated at 60° C. for 30 minutes. The reaction mixture was thencooled to room temperature and quenched with ether (100 mL). The etherwas decanted and the remaining solid was dissolved in MeOH. The productwas purified by preparative HPLC and converted to the HCl salt to give3-benzyloxy-7-iodosancycline as a yellow solid.

Synthesis of 3-Benzyloxy-7-(3′-trifluoromethylphenyl)sancycline

A solution of sodium carbonate (670 mg, 6.32 mmol) in water (5 mL) wasadded to a stirred suspension of 7-iodo-3-benzyloxysancycline (1.00 g,1.58 mmol) and Pd(OAc)₂ (100 mg, 0.44 mmol) in methanol (10 mL) at 60°C. under nitrogen. The resulting suspension was stirred at 60° C. for 10min. 4-Trifluoromethylphenyl boronic acid (0.6 g, 3.16 mmol) in methanol(10 mL) was then added and the reaction mixture was heated at 60° C. for3 hours under nitrogen. The warm reaction mixture was filtered andconcentrated. The crude product was purified by preparative HPLC andconverted to the HCl salt to give3-benzyloxy-7-(3′-trifluoromethylphenyl)sancycline as a pale brownsolid.

Synthesis of Ethyl (7-iodo 3-sancyclineoxy)acetate

60% NaH (121 mg, 3.04 mmol) was added in small portions to a stirredsolution of 7-iodosancycline TFA salt (0.5 g, 0.76 mmol) in DMF (10 mL)at room temperature. The resulting suspension was stirred at roomtemperature for 5 minutes. Ethyl bromoacetate (0.252 mL, 2.28 mmol) wasadded and heated at 60° C. for 2 hours. The reaction mixture was thencooled to room temperature and quenched with ether (100 mL). The etherwas then decanted and the solid was dissolved in MeOH. The product waspurified by preparative HPLC and the resulting solid was converted toHCl salt to give ethyl (7-iodo 3-sancyclineoxy)acetate as a yellowsolid.

Synthesis of 12a-methylamino Doxycycline

2 g of Doxycycline was dissolved in 10 ml of thionyl chloride. Thereaction mixture was heated at 55 C for ˜2 hrs. The solution was cooledin an ice-bath and then added slowly to ice-cold ether. Brownish solidprecipitated out which was filtered and dried.

300 mg of SOCl₂-doxcycline complex was dissolved in 3 ml of 2M solutionof methylamine in methanol. The starting material disappeared rapidlyand a new peak appeared with an M+H of 427. As the reaction proceeded,this material converted to a new peak with lower retention time and themass showed 440. This product was isolated by preprative HPLC andidentified by 2D NMR to be the product, exhibiting characteristic NMRpeaks and NOE interactions.

Example 2 In Vitro Minimum Inhibitory Concentration (MIC) Assay

The following assay is used to determine the efficacy of tetracyclinecompounds against common bacteria. 2 mg of each compound is dissolved in100 μl of DMSO. The solution is then added to cation-adjusted MuellerHinton broth (CAMHB), which results in a final compound concentration of200 μg per ml. The tetracycline compound solutions are diluted to 50 μLvolumes, with a test compound concentration of 0.098 μg/ml. Opticaldensity (OD) determinations are made from fresh log-phase broth culturesof the test strains. Dilutions are made to achieve a final cell densityof 1×10⁶ CFU/ml. At OD=1, cell densities for different genera should beapproximately:

E. coli   1 × 10⁹ CFU/ml S. aureus   5 × 10⁸ CFU/ml Enterococcus sp. 2.5× 10⁹ CFU/ml

50 μl of the cell suspensions are added to each well of microtiterplates. The final cell density should be approximately 5×10⁵ CFU/ml.These plates are incubated at 35° C. in an ambient air incubator forapproximately 18 hr. The plates are read with a microplate reader andare visually inspected when necessary. The MIC is defined as the lowestconcentration of the tetracycline compound that inhibits growth.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, numerous equivalents to thespecific procedures described herein. Such equivalents are considered tobe within the scope of the present invention and are covered by thefollowing claims. The contents of all references, patents, and patentapplications cited throughout this application are hereby incorporatedby reference. The appropriate components, processes, and methods ofthose patents, applications and other documents may be selected for thepresent invention and embodiments thereof.

1. A compound of Formula I:

wherein: X is CHC(R¹³Y′Y) or CR^(6′)R⁶; R^(2″) is —C(═O)NR²R^(2′) or—CN; R², R^(2′), R^(4a), and R^(4b) are each independently hydrogen,alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylsulfinyl,alkylsulfonyl, alkylamino, arylalkyl, aryl, heterocyclic, heteroaromaticor a prodrug moiety; R³, R¹⁰, R¹¹, R¹², and R^(12″) are eachindependently hydrogen, alkyl, alkenyl, aryl, alkynyl, arylalkyl,acetyl, alkylcarbonyl, alkenylcarbonyl, arylcarbonyl, alkynylcarbonyl,alkyloxycarbonyl, alkenyloxycarbonyl, alkynyloxycarbonyl,aryloxycarbonyl, alkylaminocarbonyl, alkenylaminocarbonyl,alkynylaminocarbonyl, arylaminocarbonyl, alkylthiocarbonyl,alkenylthiocarbonyl, alkynylthiocarbonyl, arylthiocarbonyl,alkyloxythiocarbonyl, alkenyloxythiocarbonyl, alkynyloxythiocarbonyl,aryloxythiocarbonyl, alkylaminothiocarbonyl, alkenylaminothiocarbonyl,alkynylaminothiocarbonyl, arylaminothiocarbonyl, alkylthiothiocarbonyl,alkenylthiothiocarbonyl, alkynylthiothiocarbonyl, arylthiothiocarbonyl,provided that at least one of R³, R¹⁰, R¹¹, or R¹² is not hydrogen whenR^(2″) is —C(═O)NR²R^(2′) and R^(12′) is OR¹²; R⁴ and R^(4′) are eachindependently NR^(4a)R^(4b), alkyl, alkenyl, alkynyl, hydroxyl, halogen,hydrogen, or when taken together the oxygen of a carbonyl group; R⁵ ishydroxyl, hydrogen, thiol, alkanoyl, aroyl, alkaroyl, aryl,heteroaromatic, alkyl, alkenyl, alkynyl, alkoxy, alkylthio,alkylsulfinyl, alkylsulfonyl, alkylamino, arylalkyl, alkyl carbonyloxy,or aryl carbonyloxy; R⁶ and R^(6′) are each independently hydrogen,methylene, absent, hydroxyl, halogen, thiol, alkyl, alkenyl, alkynyl,aryl, alkoxy, alkylthio, alkylsulfinyl, alkylsulfonyl, alkylamino, orarylalkyl; R⁷ is amino; R⁸ is hydrogen; R⁹ is hydrogen; R^(12′) is OR¹²or NR¹²R^(12″); R¹³ is hydrogen, hydroxy, alkyl, alkenyl, alkynyl,alkoxy, alkylthio, aryl, alkylsulfinyl, alkylsulfonyl, alkylamino, orarylalkyl; Y′ and Y are each independently hydrogen, halogen, hydroxyl,cyano, sulfhydryl, amino, alkyl, alkenyl, alkynyl, alkoxy, alkylthio,alkylsulfinyl, alkylsulfonyl, alkylamino, or arylalkyl; or apharmaceutically acceptable salt, ester or enantiomer thereof.
 2. Thecompound of claim 1, wherein X is CR⁶R^(6′); R², R^(2′), R⁶, R^(6′), R⁸and R¹¹ are each hydrogen; and R⁵ is hydroxyl or hydrogen.
 3. Thecompound of claim 2, wherein R⁴ dialkylamino, and R^(4′) and R⁵ are eachhydrogen.
 4. The compound of any one of claim 1, wherein R³ is alkyl,alkenyl, aryl, acetyl, arylalkyl, alkynyl, alkylcarbonyl,alkenylcarbonyl, arylcarbonyl, alkynylcarbonyl, alkyloxycarbonyl,alkenyloxycarbonyl, alkynyloxycarbonyl, aryloxycarbonyl,alkylaminocarbonyl, alkenylaminocarbonyl, alkynylaminocarbonyl,arylaminocarbonyl, alkylthiocarbonyl, alkenylthiocarbonyl,alkynylthiocarbonyl, arylthiocarbonyl, alkyloxythiocarbonyl,alkenyloxythiocarbonyl, alkynyloxythiocarbonyl, aryloxythiocarbonyl,alkylaminothiocarbonyl, alkenylaminothiocarbonyl,alkynylaminothiocarbonyl, arylaminothiocarbonyl, alkylthiothiocarbonyl,alkenylthiothiocarbonyl, alkynylthiothiocarbonyl, orarylthiothiocarbonyl.
 5. The compound of claim 4, wherein R³ isarylalkyl.
 6. The compound of claim 5, wherein R³ is benzyl.
 7. Thecompound of claim 4, wherein R³ is alkyl.
 8. The compound of claim 4,wherein R³ is acetyl.
 9. The compound of claim 4, wherein R³ is alkenyl.10. The compound of claim 1, wherein R³ is hydrogen.
 11. The compound ofclaim 1, wherein R¹⁰ is alkyl, alkenyl, aryl, acetyl, arylalkyl,alkynyl, alkylcarbonyl, alkenylcarbonyl, arylcarbonyl, alkynylcarbonyl,alkyloxycarbonyl, alkenyloxycarbonyl, alkynyloxycarbonyl,aryloxycarbonyl, alkylaminocarbonyl, alkenylaminocarbonyl,alkynylaminocarbonyl, arylaminocarbonyl, alkylthiocarbonyl,alkenylthiocarbonyl, alkynylthiocarbonyl, arylthiocarbonyl,alkyloxythiocarbonyl, alkenyloxythiocarbonyl, alkynyloxythiocarbonyl,aryloxythiocarbonyl, alkylaminothiocarbonyl, alkenylaminothiocarbonyl,alkynylaminothiocarbonyl, arylaminothiocarbonyl, alkylthiothiocarbonyl,alkenylthiothiocarbonyl, alkynylthiothiocarbonyl, orarylthiothiocarbonyl.
 12. The compound of claim 11, wherein R¹⁰ isarylalkyl.
 13. The compound of claim 12, wherein R¹⁰ is benzyl.
 14. Thecompound of claim 11, wherein R¹⁰ is alkyl.
 15. The compound of claim11, wherein R¹⁰ is acetyl.
 16. The compound of claim 11, wherein R¹⁰ isalkenyl.
 17. The compound of claim 1, wherein R¹⁰ is hydrogen.
 18. Thecompound of claim 1, wherein R¹² is alkyl, alkenyl, aryl, acetyl,arylalkyl, alkynyl, alkylcarbonyl, alkenylcarbonyl, arylcarbonyl,alkynylcarbonyl, alkyloxycarbonyl, alkenyloxycarbonyl,alkynyloxycarbonyl, aryloxycarbonyl, alkylaminocarbonyl,alkenylaminocarbonyl, alkynylaminocarbonyl, arylaminocarbonyl,alkylthiocarbonyl, alkenylthiocarbonyl, alkynylthiocarbonyl,arylthiocarbonyl, alkyloxythiocarbonyl, alkenyloxythiocarbonyl,alkynyloxythiocarbonyl, aryloxythiocarbonyl, alkylaminothiocarbonyl,alkenylaminothiocarbonyl, alkynylaminothiocarbonyl,arylaminothiocarbonyl, alkylthiothiocarbonyl, alkenylthiothiocarbonyl,alkynylthiothiocarbonyl, or arylthiothiocarbonyl.
 19. The compound ofclaim 18, wherein R¹² is arylalkyl.
 20. The compound of claim 19,wherein R¹² is benzyl.
 21. The compound of claim 18, wherein R¹² isalkyl.
 22. The compound of claim 18, wherein R¹² is acetyl.
 23. Thecompound of claim 18, wherein R¹² is alkenyl.
 24. The compound of claim1, wherein R¹² is hydrogen.
 25. A compound selected from the groupconsisting of:


26. A pharmaceutical composition comprising a therapeutically effectiveamount of a compound of claim 1 or 25, and a pharmaceutically acceptablecarrier.
 27. The compound of claim 1, wherein said compound is:


28. A compound of Formula I:

wherein: X is CHC(R¹³Y′Y), or CR^(6′)R⁶; R^(2″) is —C(O)NR²R^(2′); R²,R^(2′), R^(4a), and R^(4b) are each independently hydrogen, alkyl,alkenyl, alkynyl, alkoxy, alkylthio, alkylsulfinyl, alkylsulfonyl,alkylamino, arylalkyl, aryl, heterocyclic, heteroaromatic or a prodrugmoiety; R¹⁰ is alkyl, alkenyl, aryl, alkynyl, arylalkyl, acetyl,alkylcarbonyl, alkenylcarbonyl, arylcarbonyl, alkynylcarbonyl,alkyloxycarbonyl, alkenyloxycarbonyl, alkynyloxycarbonyl,aryloxycarbonyl, alkylaminocarbonyl, alkenylaminocarbonyl,alkynylaminocarbonyl, arylaminocarbonyl, alkylthiocarbonyl,alkenylthiocarbonyl, alkynylthiocarbonyl, arylthiocarbonyl,alkyloxythiocarbonyl, alkenyloxythiocarbonyl, alkynyloxythiocarbonyl,aryloxythiocarbonyl, alkylaminothiocarbonyl, alkenylaminothiocarbonyl,alkynylaminothiocarbonyl, arylaminothiocarbonyl, alkylthiothiocarbonyl,alkenylthiothiocarbonyl, alkynylthiothiocarbonyl, orarylthiothiocarbonyl; R³, R¹¹, R¹², and R^(12″) are each hydrogen; R⁴and R^(4′) are each independently NR^(4a)R^(4b), alkyl, alkenyl,alkynyl, hydroxyl, halogen, hydrogen, or when taken together the oxygenof a carbonyl group; R⁵ is hydroxyl, hydrogen, thiol, alkanoyl, aroyl,alkaroyl, aryl, heteroaromatic, alkyl, alkenyl, alkynyl, alkoxy,alkylthio, alkylsulfinyl, alkylsulfonyl, alkylamino, arylalkyl, alkylcarbonyloxy, or aryl carbonyloxy; R⁶ and R^(6′) are each independentlyhydrogen, methylene, absent, hydroxyl, halogen, thiol, alkyl, alkenyl,alkynyl, aryl, alkoxy, alkylthio, alkylsulfinyl, alkylsulfonyl,alkylamino, or arylalkyl; R⁷ is amino; R⁸ is hydrogen; R⁹ is hydrogen;R^(12′) is OR¹² or NR¹²R^(12″); R¹³ is hydrogen, hydroxy, alkyl,alkenyl, alkynyl, alkoxy, alkylthio, aryl, alkylsulfinyl, alkylsulfonyl,alkylamino, or arylalkyl; Y′ and Y are each independently hydrogen,halogen, hydroxyl, cyano, sulfhydryl, amino, alkyl, alkenyl, alkynyl,alkoxy, alkylthio, alkylsulfinyl, alkylsulfonyl, alkylamino, orarylalkyl; or a pharmaceutically acceptable salt, ester or enantiomerthereof.